Optimization Of Preparation Process For Portulaca Oleracea L.polysaccharide Liposomes And Preliminary Evaluation Of Its Immune Enhancement Activity

Objective:Portulaca oleracea L.（POL）is a widely distributed Chinese herbal medicine with food homology,known for its functions in clearing heat,detoxifying,and regulating immunity,among others.In recent years,POL-Polysaccharide（POL-P）has been extracted,isolated,and purified from POL.Previous studies have indicated that POL-P can enhance the body’s immunity and reduce toxins.However,it has been observed that POL-P’s immune-boosting effects exhibit certain limitations,such as low bioavailability and inadequate research in conjunction with new drug delivery systems,resulting in restricted applications of POL-P.Liposomes are aqueous core micro-vesicles enveloped by a phospholipid bilayer.As an outstanding drug carrier,liposomes can enhance bioavailability while reducing toxicity and side effects.In this study,we aim to combine the immune-enhancing effects of POL-P with the favorable properties of liposomes as drug carriers and establish the optimal preparation process for POL-P liposome（POL-PL）and conduct a preliminary evaluation of its immune-enhancing activity.By combining the actions of POL-P liposome and POL-PL,we seek to address the limitations of POL-P application and further enhance its immune-boosting activity.Methods:First,the optimum preparation process of POL-PL was obtained by single factor experiment and response surface optimization.POL-PL was prepared by reverse-phase evaporation-microporous filtration membrane extrusion method.Based on the single-factor experiment with six factors and five levels,the response surface optimization carried out three single-factor conditions that better influence the encapsulation rate,namely,evaporation temperature,drug-loading capacity,and membrane material ratio.The other factors selected the optimal conditions.Based on the single-factor experiment with six factors and five levels,the response surface optimization carried out three single-factor conditions that better influence the encapsulation rate,namely,evaporation temperature,loading ratio,and membrane material ratio.The other factors selected the optimal conditions,then feasibility was adjusted and verified,and obtain the best preparation conditions for POL-PL were determined.Secondly,Secondly,the morphology and physicochemical properties of POL-PL prepared by the optimal technology were analyzed.The morphology and physicochemical properties of POL-PL were determined by morphological observation,transmission electron microscopy（TEM）,particle size distribution,Polydispersity（PDI）and Zeta potential measurement,infrared absorption spectrum（IR）analysis,sustained release,and storage stability tests.Further using the method of CCK 8 will POL-P,Blank liposomes（BL）,Thirdly,the immuno-enhancement activity of POL-PL on mouse splenic lymphocytes was preliminarily evaluated.CCK-8 method was used to investigate the maximum safe concentration of POL-P,BL,and POL-PL on spleen lymphocytes of mice,and the optimal concentration was selected to detect the effects of POL-PL on spleen lymphocytes of mice,the proliferation of T cells,IgG secretion of B lymphocytes and differentiation of T lymphocytes.To determine the preliminary evaluation of POL-PL in enhancing the immune activity of spleen lymphocytes in mice Finally,the immuno-enhancement activity of POL-PL against mouse macrophage Raw264.7 was evaluated.The maximum safe concentration of POL-PL on mouse macrophage Raw264.7 was determined by CCK-8 method,and the optimal concentration was used to detect the proliferation and phagocytosis activity of Raw264.7 cells,and the cytokines including Tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）,interleukin-6（IL-6）,and Nitric oxide（NO）levels,POL-PL showed a preliminary evaluation of splenic lymphocyte immune-enhancing activity in mice.Results:1.The optimum preparation conditions of POL-PL are as follows:the membrane material ratio is 5:1;the Rotary steaming temperature is 40℃;The ratio of lipid to the drug was 27:1.The encapsulation rate was 37.83±1.49%,which was consistent with the model prediction.2.POL-PL was transparent and bright with light blue opalescence.Under the TEM,POL-PL was a spherical or elliptic vesicle.The average particle size and PDI were 100.037±1.990nm and0.232±0.003,respectively,and Zeta potential was（-23.033±1.686）m V（n=3）.Infrared results show that POL-PL does not form new chemical bonds during its formation,but is formed by electrostatic force.POL-PL has good sustained-release properties,and its encapsulation rate decreases by 5%when stored at 4℃for one month.3.The maximum safe concentration of POL-PL for mouse lymphocytes were found to be62.5μg·m L^-1,with the optimal training time being 24 h.When compared to the POL-P,BL,and BC groups,POL-PL significantly promoted the proliferation of mouse spleen B lymphocytes and T cells（P<0.05）,with the most effective dose being 16μg·m L^-1.Furthermore,POL-PL notably increased the secretion of splenic B cell IgG（P<0.05）.Flow cytometry analysis revealed that the ratio of CD4⁺/CD8⁺to splenic T-cell subsets in mice was the most significant（P<0.05）.4.The maximum safe concentration of POL-PL for mice macrophage Raw264.7 were found to be 62.5μg·m L^-1,with the optimal training time being 24 h.When compared to the POL-P,BL,and BC groups,POL-PL significantly promoted the proliferation and phagocytosis activity of Raw264.7 cells（P<0.05）,with the most effective dose being 16μg·m L^-1.Furthermore,POL-PL significantly increased the levels of TNF-α,IL-1β,IL-6,and NO secreted by Raw264.7 cells（P<0.05）.Conclusion:In this study,POL-PL was prepared by reverse phase evaporation-microporous filtration membrane extrusion method,and the single factor-response surface method was used to optimize the optimum preparation process.The morphology and physicochemical properties of POL-PL were analyzed according to the optimal preparation process,and further in vitro experiments were conducted to confirm the sustained release of POL-P after being coated with liposome,and to verify the immune enhancement activity of POL-P on mouse spleen lymphocytes and macrophage Raw264.7.The results of this study provide ideas for improving the active ingredients of traditional Chinese medicine and theoretical basis for the research and development of polysaccharide immune enhancers.